Antibodies specific for ELAM-1 and the use thereof

ABSTRACT

The invention relates to a method for detecting ELAM-1 expression in a patient comprising (i) administration of a detectable amount of a labelled ELAM-1 specific antibody (or an anti-ELAM 1 antibody fragment) and a pharmaceutically acceptable carrier, and (ii) detecting the label. ELAM-1 is an endothelial cell surface glycoprotein which participates in endothelialleukocyte adhesion. Induction of ELAM-1 expression has been detected on endothelial cells at sites of inflammation.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. HL 36028awarded by the National Institutes of Health. The government has certainrights in the invention.

This application is a continuation of application Ser. No. 07/850,802,filed Mar. 13, 1992, (now abandoned), which is a division of applicationSer. No. 07,270,860, filed Nov. 14, 1988, (now abandoned).

BACKGROUND OF THE INVENTION

Focal adhesion of leukocytes to the endothelial lining of blood vesselsis a characteristic step in inflammation and in certain vascular diseaseprocesses. Although it has long been suspected that alterations in thevessel wall could promote leukocyte adhesion (Cohnheim, J., Lectures inGeneral Pathology. (New Syndenham Society, London) Vol. 1, 2nd Ed.(1898)), until recently, potential mechanisms and modulators of thisinteraction have remained undefined (Harlan, J. M., Blood. 65:513-525(1985)). Certain inflammatory/immune cytokines including interleukin-1(IL-1) and tumor necrosis factor (TNF), as well as bacterial endotoxin,have been shown to act directly on cultured human vascular endotheliumto increase the adhesiveness of its surface for blood leukocytes andrelated cell lines. Bevilacqua, M. P. et al., J. Clin. Invest.76:2003-2011 (1985); Bevilacqua, M. P. et al., Am. J. Pathol.121:393-403 (1985); Bevilacqua, M. P. et al., Leukocyte Emigration andIts Sequelae, Movat, H. Z. (ed.), Karger, N.Y., pp. 79-93 (1987);Gamble, J. R. et al., Proc. Natl. Acad. Sci. teu(USA ) 82:8667-8671(1985); Schleimer, R. P. et al., J. Immunol. 136:649-654 (1986); Dunn,C. J. et al., The Physiologic, Metabolic and Immunologic Actions ofInterleukin-1, Kluger, M. J. et al., (eds.), Liss, N.Y. pp. 45-54(1985); Cavender, D. E. et al., J. Immunol. 136:203-207 (1986); Yu,C.-L. et al., J. Immunol. 136:571 (1986); and Pohlman, T. H. et al., J.Immunol. 136:4548-4553 (1986).

A number of leukocyte and endothelial cell surface proteins participatein endothelial cell-leukocyte adhesion. One example, ICAM-1, isexpressed on a variety of hemoporetic cells and non-hemopoietic cellsincluding vascular endothelium. ICAM-1 is a 90 kD glycoprotein which isinduced by certain cytokines on endothelial cells. ICAM-1 expression isprotein- and mRNA sythesis-dependent and its induction is reversible.Dustin, M. L., et al., J. Immunol. 137:245-254 (1986); Rothlein, R., etal., J. Immunol. 137:1270-1274 (1986); and Pober, J. S., et al., J.Immunol. 136:1680-1687 (1986).

A leukocyte cell surface glycoprotein, LFA-1, is expressed onlymphocytes, granulocytes, monocytes, phagocytes and B cells. LFA-1 alsoplays a role in leukocyte-endothelial cell adhesion. LFA-1 is a dimericmolecule with a 177 kD (alpha or heavy) subunit and a 95 kD (beta orlight) subunit. An F(ab')2 Mab has been developed to the alpha and betasubunits which effectively inhibits leukocyte adhesion to endothelialcells. Mentzer, S. J. et al., J. Cell. Physiol. 126:285-290 (1986);Mentzer, S. J. et al., J. Cell. Physiol. 130:410-415 (1987); andPohlman, J. H., et al., J. Immunol. 136:4548-4553 (1986).

FIELD OF THE INVENTION

The invention is in the field of immunology.

SUMMARY OF THE INVENTION

The invention relates to a cloned gene or degenerate variant thereof,which encodes ELAM-1 or a fragment thereof.

The cloned gene of the invention comprises the following DNA sequence:

    __________________________________________________________________________      1                                                                              CCTGAGACAG                                                                             AGGCAGCAGT                                                                              GATACCCACC                                                                              TGAGAGATCC                                                                              TGTGTTTGAA                           51                                                                              CAACTGCTTC                                                                             CCAAAACGGA                                                                              AAGTATTTCA                                                                              AGCCTAAACC                                                                              TTTGGGTGAA                           101                                                                             AAGAACTCTT                                                                             GAAGTCATGA                                                                              TTGCTTCACA                                                                              GTTTCTCTCA                                                                              GCTCTCACTT                           151                                                                             TGGTGCTTCT                                                                             CATTAAAGAG                                                                              AGTGGAGCCT                                                                              GGTCTTACAA                                                                              CACCTCCACG                           201                                                                             GAAGCTATGA                                                                             CTTATGATGA                                                                              GGCCAGTGCT                                                                              TATTGTCAGC                                                                              AAAGGTACAC                           251                                                                             ACACCTGGTT                                                                             GCAATTCAAA                                                                              ACAAAGAAGA                                                                              GATTGAGTAC                                                                              CTAAACTCCA                           301                                                                             TATTGAGCTA                                                                             TTCACCAACT                                                                              TATTACTGGA                                                                              TTCGAATCAG                                                                              AAAAGTCAAC                           351                                                                             AATGTGTGGG                                                                             TCTGGGTAGG                                                                              AACCCAGAAA                                                                              CCTCTGACAG                                                                              AAGAAGCCAA                           401                                                                             GAACTGCGCT                                                                             CCAGGTGAAC                                                                              CCAACAATAG                                                                              GCAAAAAGAT                                                                              GAGGACTGCG                           451                                                                             TGGAGATCTA                                                                             CATCAAGAGA                                                                              GAAAAAGATG                                                                              TGGGCATGTG                                                                              CAATGATGAG                           501                                                                             AGGTGCACCA                                                                             AGAAGAAGCT                                                                              TGCCCTATGC                                                                              TACACAGCTG                                                                              CCTGTACCAA                           551                                                                             TACATCCTGC                                                                             AGTGCCCACG                                                                              GTGAATGTGT                                                                              AGAGACCATC                                                                              AATAATTACA                           601                                                                             CTTGCAACTG                                                                             TGACCCTGCC                                                                              TTCAGTGCAC                                                                              TCAAGTGTGA                                                                              GCAAATTGTG                           651                                                                             AACTGTACAG                                                                             CCCTGGAATC                                                                              CCCTGAGCAT                                                                              GGAAGCCTGG                                                                              TTTGCAGTCA                           701                                                                             CCCACTGGGA                                                                             AACTTCAGCT                                                                              ACAATTCTTC                                                                              CTGCTCTATC                                                                              AGCTGTGATA                           751                                                                             GGGGTTACCT                                                                             CCCAAGCAGC                                                                              ATGGAGACCA                                                                              TGCAGTGTAT                                                                              GTCCTCTGGA                           801                                                                             GAATGGAGTG                                                                             CTCCTATTCC                                                                              AGCCTGCAAT                                                                              GTGGTTGAGT                                                                              GTGATGCTGT                           851                                                                             CACAAATCCA                                                                             GCCAATGGGT                                                                              TCGTGGAATG                                                                              TTTCCAAAAC                                                                              CCTGGAAGCT                           901                                                                             TCCCATGGAA                                                                             CACAACCTGT                                                                              ACATTTGACT                                                                              GTGAAGAAGG                                                                              ATTTCAACTA                           951                                                                             ATCGGAGCCC                                                                             AGAGCCTTCA                                                                              GTGTACCTCA                                                                              TCTGGGAATT                                                                              GGGACAACGA                          1001                                                                             GAAGCCAACG                                                                             TGTAAAGCTC                                                                              TGACATGCAG                                                                              GGCCGTCCGC                                                                              CACCCTCAGA                          1051                                                                             ATGCCTCTGT                                                                             GAGGTGCAGC                                                                              CATTCCCCTG                                                                              CTGGAGAGTT                                                                              CACCTTCAAA                          1101                                                                             TCATCCTGCA                                                                             ACTTCACCTG                                                                              TGAGGAAGGC                                                                              TTCATGTTGC                                                                              AGGGACCAGC                          1151                                                                             CCACGTTGAA                                                                             TGCACCACTC                                                                              AAGGGCAGTG                                                                              GACACAGCAA                                                                              ATCCCAGTTT                          1201                                                                             GTGAAGCTTT                                                                             CCAGTGCACA                                                                              GCCTTGTCCA                                                                              ACCCCCACCC                                                                              AGGCTACATC                          1251                                                                             AATTGTCTTC                                                                             CTAGTGCTTC                                                                              TGGCAGTTTC                                                                              CGTTATGGGT                                                                              CCAGCTCTGA                          1301                                                                             GTTCTCCTGT                                                                             CACCAGGGTT                                                                              TTGTGTTGAA                                                                              GCCATCCAAA                                                                              AGGCTCCAAT                          1351                                                                             GTGGCCCCAC                                                                             AGGGGAGTGG                                                                              GACAACGAGA                                                                              AGCCCACATG                                                                              TCAAGCTGTG                          1401                                                                             AGATGCGATG                                                                             CTGTCCACCA                                                                              GCCCCCGAAG                                                                              GGTTTGGTGA                                                                              GGTGTGETCA                          1451                                                                             TTCCCCTATT                                                                             CGAGAATTCA                                                                              CCTACAAGTC                                                                              CTCTTGTGCC                                                                              TTCAGCTGTG                          1501                                                                             ACCAGGCATT                                                                             TCAATTATAT                                                                              GCATCAACTC                                                                              AACTTGACTG                                                                              CACATCTCAG                          1551                                                                             GGACAATCCA                                                                             CAGAAGAGCT                                                                              TCCTTCCTCC                                                                              CAACTGCTAA                                                                              AATGTTCAAC                          1601                                                                             CCTGGCAGTT                                                                             CCGGGAAAGA                                                                              TCAACATGAG                                                                              CTGCAGTGGG                                                                              GAGCCCGTGT                          1651                                                                             TTCGCACTGT                                                                             GTGCAAGTTC                                                                              GCCTGTCCTG                                                                              AACCATGCAC                                                                              GCTCAATGGC                          1701                                                                             TCTGCAGCTC                                                                             GGACATGTGG                                                                              ACCCACAGGA                                                                              CACTGCTCTG                                                                              GCCTGCTACC                          1751                                                                             TACCTCTCAA                                                                             CCTCCCACTG                                                                              ACTCCAACAT                                                                              TCCCTTGCTA                                                                              CCTGCACTTT                          1801                                                                             CTGCTGCTGG                                                                             ACTCTCCCTC                                                                              CTGACATTAG                                                                              CACCATTTCT                                                                              CCTCTCGCTT                          1851                                                                             CGGAAATGCT                                                                             TACGGAAACC                                                                              AAAGAAATTT                                                                              GTTCCTGCCA                                                                              GCAGCTGCCA                          1901                                                                             AACCCTTGAA                                                                             TCAGACGGAA                                                                              GCTACCAAAA                                                                              GCCTTCTTAC                                                                              ATCCTTTAAG                          1951                                                                             TTCAAAAGAA                                                                             TCAGAAACAG                                                                              GTGCATCTGG                                                                              CCAACTAGAG                                                                              GGATACACTG                          2001                                                                             AAGTTAACAG                                                                             AGACAGATAA                                                                              CTCTCCTCGG                                                                              GTCTCTGGCC                                                                              CTTCTTGCCT                          2051                                                                             ACTATGCCAG                                                                             ATCCCTTTAT                                                                              CGCTGAAACC                                                                              CCAACACCCA                                                                              TCACCACTTC                          2101                                                                             AATAGATCAA                                                                             AGTCCAGCAG                                                                              GCAAGGACGG                                                                              CCTTCAACTG                                                                              AAAAGACTCA                          2151                                                                             GTGTTCCCTT                                                                             TCCTACTCTC                                                                              AGGATCAAGA                                                                              AAGTGTTGGC                                                                              TAATGAAGGG                          2201                                                                             AAAGGATATT                                                                             TTCTTCCAAG                                                                              CAAAGGTGAA                                                                              AACACCAACA                                                                              CTCTCAAATC                          2251                                                                             TCAGAATTCC                                                                             TTTTCTAACT                                                                              CTCCCTTGCT                                                                              CGCTGTAAAA                                                                              TCTTGGCACA                          2301                                                                             CAAACACAAT                                                                             ATTTTGTGGC                                                                              TTTCTTTCTT                                                                              TTGCCCTTCA                                                                              CAGTGTTTCG                          2351                                                                             ACAGCTGATT                                                                             ACACAGTTGC                                                                              TGTCATAAGA                                                                              ATGAATAATA                                                                              ATTATCCAGA                          2401                                                                             GTTTACAGGA                                                                             AAAAAATGAC                                                                              TAAAAATATT                                                                              ATAACTTAAA                                                                              AAAATGACAG                          2451                                                                             ATGTTGAATG                                                                             CCCACAGGCA                                                                              AATGCATGGA                                                                              GGCTTGTTAA                                                                              TGGTGCAAAT                          2501                                                                             CCTACTGAAT                                                                             GCTCTGTGCG                                                                              AGGGTTACTA                                                                              TGCACAATTT                                                                              AATCACTTTC                          2551                                                                             ATCCCTATGC                                                                             CATTCAGTGC                                                                              TTCTTAAAGA                                                                              GTTCTTAACC                                                                              ATTGTGATAT                          2601                                                                             TTTTACTTGC                                                                             ATTGAATATA                                                                              TTATAATCTR                                                                              CCATACTTCT                                                                              TCATTCAATA                          2651                                                                             CAAGTGTGGT                                                                             ACGCACTTAA                                                                              AAAACTTCTA                                                                              AATGCTGTCA                                                                              ACTATGATAT                          2701                                                                             GGTAAAAGTT                                                                             ACTTATTCTA                                                                              GATTACCCCC                                                                              TCATTGTTTA                                                                              TTAACAAATT                          2751                                                                             ATGTTACATC                                                                             TGTTTTAAAT                                                                              TTATTTCAAA                                                                              AAGGGAAACT                                                                              ATTGTCCCCT                          2801                                                                             AGCAAGGCAT                                                                             GATGTTAACC                                                                              AGAATAAAGT                                                                              TCTGAGTGTT                                                                              TTTACTACAG                          2851                                                                             TTGTTTTTTC                                                                             AAAACATGGT                                                                              AGAATTGGAG                                                                              AGTAAAAACT                                                                              GAATGGAAGG                          2901                                                                             TTTGTATATT                                                                             GTCAGATATT                                                                              TTTTCAGAAA                                                                              TATGTGGTTT                                                                              CCACGATGAA                          2951                                                                             AAACTTCCAT                                                                             GAGGCCAAAC                                                                              GTTTTGAACT                                                                              AATAAAAGCA                                                                              TAAATGCAAA                          3001                                                                             CACACAAAGG                                                                             TATAATTTTA                                                                              TGAATCTCTT                                                                              TGTTGGAAAA                                                                              GAATACAGAA                          3061                                                                             AGATGGATGT                                                                             GCTTTGCATT                                                                              CCTACAAAGA                                                                              TGTTTGTCAG                                                                              ATGTGATATG                          3101                                                                             TAAACATAAT                                                                             TCTTGTATAT                                                                              TATGGAAGAT                                                                              TTTAAATTCA                                                                              CAATAGAAAC                          3151                                                                             TCACCATGTA                                                                             AAAGAGTCAT                                                                              CTGGTACATT                                                                              TTTAACGAAT                                                                              GAAGATGTCT                          3201                                                                             AATAGTTATT                                                                             CCCTATTTGT                                                                              TTTCTTCTGT                                                                              ATGTTAGGGT                                                                              GCTCTGGAAG                          3251                                                                             AGAGGAATGC                                                                             CTGTGTGAGC                                                                              AAGCATTTAT                                                                              GTTTATTTAT                                                                              AAGCAGATTT                          3301                                                                             AACAATTCCA                                                                             AAGGAATCTC                                                                              CAGTTTTCAG                                                                              TTCATCACTG                                                                              GCAATGAAAA                          3351                                                                             ATTCTCAGTC                                                                             AGTAATTGCC                                                                              AAAGCTGCTC                                                                              TAGCCTTGAG                                                                              GAGTGTGAGA                          3401                                                                             ATCAAAACTC                                                                             TCCTACACTT                                                                              CCATTAACTT                                                                              AGCATGTGTT                                                                              GAAAAAAAAA                          3451                                                                             GTTTCAGAGA                                                                             AGTTCTGGCT                                                                              GAACACTGGC                                                                              AACGACAAAG                                                                              CCAACAGTCA                          3501                                                                             AAACAGAGAT                                                                             GTGATAAGGA                                                                              TCAGAACAGC                                                                              AGAGGTTCTT                                                                              TTAAAGGGGC                          3551                                                                             AGAAAAACTC                                                                             TGGGAAATAA                                                                              GAGAGAACAA                                                                              CTACTGTGAT                                                                              CAGGCTATGT                          3601                                                                             ATGGAATACA                                                                             GTGTTATTTT                                                                              CRTTGAAATT                                                                              GTTTAAGTGT                                                                              TGTAAATATT                          3651                                                                             TATGTAAACT                                                                             GCATTAGAAA                                                                              TTAGCTGTGT                                                                              GAAATACCAG                                                                              TGTGGTTTCT                          3701                                                                             GTTTGAGTTT                                                                             TATTGAGAAT                                                                              TTTAAATTAT                                                                              AACTTAAAAT                                                                              ATTTTATAAT                          3751                                                                             TTTTAAAGTA                                                                             TATATTTATT                                                                              TAAGCTTATG                                                                              TCAGACCTAT                                                                              TTGACATAAC                          3801                                                                             ACTATAAAGG                                                                             TTGACAATAA                                                                              ATGTGCTTAT                                                                              GTTTAAAAAA                                                                              AAAAAAAAAA                          3851                                                                             AAAA                                                                       __________________________________________________________________________

The invention also relates to a cloned gene comprising the ELAM-1 codingsequence having the following DNA sequence:

    __________________________________________________________________________                ATGA      TTGCTTCACA                                                                              GTTTCTCTCA                                                                              GCTCTCACTT                           151                                                                             TCGTGCTTCT                                                                             CATTAAAGAC                                                                              AGTGGAGCCT                                                                              GGTCTTACAA                                                                              CACCTCCACG                           201                                                                             GAAGCTATGA                                                                             CTTATGATGA                                                                              GGCCAGTGCT                                                                              TATTGTCAGC                                                                              AAAGGTACAC                           251                                                                             ACACCTGGTT                                                                             GCAATTCAAA                                                                              ACAAAGAAGA                                                                              GATTGAGTAC                                                                              CTAAACTCCA                           301                                                                             TATTGAGCTA                                                                             TTCACCAAGT                                                                              TATTACTGGA                                                                              TTGGAATCAG                                                                              AAAAGTCAAC                           351                                                                             AATGTGTGGG                                                                             TCTGGGTAGG                                                                              AACCCAGAAA                                                                              CCTCTGACAG                                                                              AAGAAGCCAA                           401                                                                             GAACTGGGCT                                                                             CCAGGTGAAC                                                                              CCAACAATAG                                                                              GCAAAAAGAT                                                                              GAGGACTGCG                           451                                                                             TGGAGATCTA                                                                             CATCAAGAGA                                                                              GAAAAAGATG                                                                              TGGGCATGTG                                                                              GAATGATGAG                           501                                                                             AGGTGCAGCA                                                                             AGAAGAAGCT                                                                              TGCCCTATGC                                                                              TACACAGCTG                                                                              CCTGTACCAA                           551                                                                             TACATCCTGC                                                                             AGTGGCCACG                                                                              GTGAATGTGT                                                                              AGAGACCATC                                                                              AATAATTACA                           601                                                                             CTTGCAAGTG                                                                             TGACCCTGGC                                                                              TTCAGTGGAC                                                                              TCAAGTGTGA                                                                              GCAAATTGTG                           651                                                                             AACTGTACAG                                                                             CCCTGGAATC                                                                              CCCTGAGCAT                                                                              GGAAGCCTGG                                                                              TTTGCAGTCA                           701                                                                             CCCACTGGGA                                                                             AACTTCAGCT                                                                              ACAATTCTTC                                                                              CTGCTCTATC                                                                              AGCTGTGATA                           751                                                                             GGGGTTACCT                                                                             GCCAAGCAGC                                                                              ATGGAGACCA                                                                              TGCAGTGTAT                                                                              GTCCTCTGGA                           801                                                                             GAATGGAGTG                                                                             CTCCTATTCC                                                                              AGCCTGCAAT                                                                              GTGGTTGAGT                                                                              GTGATGCTGT                           851                                                                             GACAAATCCA                                                                             GCCAATGGGT                                                                              TCGTGGAATG                                                                              TTTCCAAAAC                                                                              CCTGGAAGCT                           901                                                                             TCCCATGGAA                                                                             CACAACCTGT                                                                              ACATTTGACT                                                                              GTGAAGAAGG                                                                              ATTTGAACTA                           951                                                                             ATGGGAGCCC                                                                             AGAGCCTTCA                                                                              GTGTACCTCA                                                                              TCTGGGAATT                                                                              GGGACAACGA                          1001                                                                             GAAGCCAACG                                                                             TGTAAAGCTC                                                                              TGACATGCAG                                                                              GGCCGTCCGC                                                                              CAGCCTCAGA                          1051                                                                             ATGGCTCTGT                                                                             GAGGTGCAGC                                                                              CATTCCCCTG                                                                              CTGGAGAGTT                                                                              CACCTTCAAA                          1101                                                                             TCATCCTGCA                                                                             ACTTCACCTG                                                                              TGAGGAAGGC                                                                              TTCATGTTGC                                                                              ACCGACCAGC                          1151                                                                             CCAGGTTGAA                                                                             TGCACCACTC                                                                              AAGGGCAGTC                                                                              GACACAGCAA                                                                              ATCCCAGTTT                          1201                                                                             GTGAAGCTTT                                                                             CCAGTGCACA                                                                              GCCTTGTCCA                                                                              ACCCCGAGCG                                                                              AGGCTACATG                          1251                                                                             AATTGTCTTC                                                                             CTAGTGCTTC                                                                              TGGCAGTTTC                                                                              CGTTATGGGT                                                                              CCAGCTGTGA                          1301                                                                             GTTCTCCTGT                                                                             GAGCAGGGTT                                                                              TTGTGTTGAA                                                                              GGGATCCAAA                                                                              AGGCTCCAAT                          1351                                                                             GTGGCCCCAC                                                                             AGGGGAGTGG                                                                              GACAACGAGA                                                                              AGCCCACATG                                                                              TGAAGCTGTG                          1401                                                                             AGATGCGATG                                                                             CTGTCCACCA                                                                              GCCCCCGAAG                                                                              GGTTTGGTGA                                                                              CCTGTGCTCA                          1451                                                                             TTCCCCTATT                                                                             GGAGAATTCA                                                                              CCTACAAGTC                                                                              CTCTTGTGCC                                                                              TTCAGCTGTG                          1501                                                                             AGGAGGGATT                                                                             TGAATTATAT                                                                              GGATCAACTC                                                                              AACTTGAGTG                                                                              CACATCTCAG                          1551                                                                             GGACAATCGA                                                                             CAGAAGAGGT                                                                              TCCTTCCTGC                                                                              CAAGTGGTAA                                                                              AATGTTCAAG                          1601                                                                             CCTGGCAGTT                                                                             CCGGGAAACA                                                                              TCAACATCAG                                                                              CTGCAGTGGG                                                                              GAGCCCGTGT                          1651                                                                             TTGGCACTGT                                                                             GTGCAAGTTC                                                                              GCCTGTCCTG                                                                              AAGGATGGAC                                                                              GCTCAATGGC                          1701                                                                             TCTGCAGCTC                                                                             GGACATGTGG                                                                              AGCCACAGGA                                                                              CACTGGTCTG                                                                              GCCTGCTACC                          1751                                                                             TACCTGTGAA                                                                             GCTCCCACTG                                                                              ACTCCAACAT                                                                              TCCCTTGGTA                                                                              GCTGGACTTT                          1801                                                                             CTGCTGCTGG                                                                             ACTCTCCCTC                                                                              CTGACATTAG                                                                              CACCATTTCT                                                                              CCTCTGGCTT                          1851                                                                             CGGAAATGCT                                                                             TACGGAAAGC                                                                              AAAGAAATTT                                                                              GTTCCTGCCA                                                                              GCAGCTGCCA                          1901                                                                             AAGCCTTGAA                                                                             TCAGACGGAA                                                                              GCTACCAAAA                                                                              GCCTTCTTAC                                                                              ATCCTTTAA                           __________________________________________________________________________

The invention also relates to ELAM-1, obtained by expressing the clonedgene of the invention, comprising the following amino acid sequence:

    __________________________________________________________________________    A S Q F L S A L T L V L L I K E S G A  W                                      S Y N T S T E A M T Y D E A S A Y C Q  Q                                      R Y T H L V A I Q N K E E I E Y L N S  I                                      L S Y S P S Y Y W I G I R K V N N V W  V                                      W V G T Q K P L T E E A K N W A P G E  P                                      N N R Q K D E D C V E I Y I K R E K D  V                                      G M W N D E R C S K K K L A L C Y T A  A                                      C T N T S C S G H G E C V E T I N N Y  T                                      C K C D P G F S G L K C E Q I V N C T  A                                      L E S P E H G S L V C S H P L G N F S  Y                                      N S S C S I S C D R G Y L P S S M E T  M                                      Q C M S S G E W S A P I P A C N V V E  C                                      D A V T N P A N G F V E C F Q N P C S  F                                      P W N T T C T F D C E E G F E L M G A  Q                                      S L Q C T S S G N W D N E K P T C K A  V                                      T C R A V R Q P Q N G S V R C S H S P  A                                      G E F T F K S S C N F T C E E G F M L  Q                                      C P A Q V E C T T Q G Q W T Q Q I P V  C                                      E A F Q C T A L S N P E R G Y M N C L  P                                      S A S G S F R Y G S S C E F S C E Q G  F                                      V L K G S K R L Q C G P T G E W D N E  K                                      P T C E A V R C D A V H Q P P K G L V  R                                      C A H S P I G E F T Y K S S C A F S C  E                                      E G F E L Y G S T Q L E C T S Q G Q W  T                                      E E V P S C Q V V K C S S L A V P G K  I                                      N M S C S G E P V F G T V C K F A C P  E                                      G W T L N G S A A R T C G A T G H W S  G                                      L L P T C E A P T E S N I P L V A G L  S                                      A A G L S L L T L A P F L L W L R K C  L                                      R K A K K F V P A S S C Q S L E S D G  S                                      Y Q K P S Y I L                                                               __________________________________________________________________________

The invention also relates to a cloned gene, or degenerate variantthereof, which encodes a fusion protein comprising ELAM-1, or a fragmentthereof, and a second protein which may be diphtheria toxin, ricin orother cytotoxic protein.

The invention also relates to plasmids containing the cloned genes ofthe invention, hosts transformed with the plasmids of the invention, andELAM-1 or a fragment thereof expressed by the transformed hosts of theinvention.

The invention also relates to fusion proteins comprising ELAM-1, or afragment thereof, and a second protein which may be diphtheria toxin,ricin or other cytotoxic protein.

The invention also relates to pharmaceutical compositions comprisingELAM-1, or a fragment thereof, and a pharmaceutically acceptablecarrier.

The invention also relates to pharmaceutical compositions comprising afusion protein comprising ELAM-1, or a fragment thereof, and a secondprotein which may be diphtheria toxin or ricin.

The invention also relates to a method for the treatment of inflammationcomprising administering to a patient a pharmaceutical compositioncomprising ELAM-1, or a fragment thereof.

The invention also relates to a method of preventing or treatingpost-reperfusion injury comprising administering to a patient apharmaceutical composition comprising ELAM-1 or a fragment thereof.

The invention also relates to a method for the treatment of leukemia orlymphoma comprising administering to a patient a pharmaceuticalcomposition comprising ELAM-1, or a fragment thereof.

The invention also relates to a treatment for infections (e.g.,bacterial, viral or parasitic) comprising administering to a patient apharmaceutical composition comprising ELAM-1, or a fragment thereof.

The invention also relates to a treatment of vasculitis comprisingadministering to a patient a pharmaceutical composition comprisingELAM-1, or a fragment thereof.

The invention also relates to a method for inhibiting metastatic spreadof tumor cells administering to a patient a pharmaceutical compositioncomprising ELAM-1 or a fragment thereof, and a pharmaceuticallyacceptable carrier.

The invention also relates to a method for the treatment of inflammationby administering to a patient a pharmaceutical composition whichcontains a therapeutically effective amount of an ELAM-1 specificantibody, or and antibody fragment thereof, and a pharmaceuticallyacceptable carrier.

The invention also relates to a method for the treatment of infection(e.g., bacterial, viral or parasitic), comprising administering to apatient a pharmaceutical composition comprising a therapeuticallyeffective amount of an ELAM-1 specific antibody, or antibody fragment,and a pharmaceutically acceptable carrier.

The invention also relates to a method for detecting ELAM-1 expressionin a patient, comprising

administering a pharmaceutical composition comprising a detectableamount of a labeled ELAM-1 specific antibody, or antibody fragment, anda pharmaceutically acceptable carrier, and

detecting the label.

The invention also relates to a method for detecting ELAM-1 expressionin a biological sample comprising contacting the sample with an ELAM-1specific antibody, or antibody fragment, and detecting whether a complexhas formed.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts an enzyme restriction map of ELAM-1 cDNA.

FIG. 2 depicts a graph showing the specific binding of H18/7 to HECmonolayers for various times after treatment with IL-1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

ELAM-1 is an endothelial cell surface glycoprotein which participates inendothelial-leukocyte adhesion. This adhesion protein is induced bycertain cytokines. ELAM-1 is identified by specific binding of murineMab (H4/18 and H18/7) developed against stimulated endothelial cells.The hybridoma producing the murine Mab H18/7 has been deposited at theAmerican Type Culture Collection (12301 Parklawn Drive, Rockville, Md.20852) on Jul. 12, 1994, under ATCC Deposit No. HB11684. The timesequence of induction of the antigen recognized by H4/18 and H18/7binding antigens and the susceptibility of its expression to metabolicinhibitors were similar to those observed for leukocyte adhesion.Bevilacqua, M. P., et al., 4th Int'l Symp. Biol. Vasc. EndothelialCell:13 (Abstr) (1986); Pober, J. S., et al., J. Immunol. 136:1680-1687(1986); Bevilacqua, M. P. et al., Proc. Natl. Acad. Sci. (USA)84:9238-9242 (1987); Bevilacqua, M. P., et al., in: LeukocyteImmigration and Its Sequelae, Satellite Symp. (Henry Z. Movat, ed.), pp.79-93, published by S. Karger, Basil, Switzerland (1987). These putativeendothelial cell surface structures were termed "endothelial-leukocyteadhesion molecules" (ELAMs). Expression of ELAM-1 by stimulatedendothelial cells was blocked by protein and RNA synthesis inhibitors,but not by cyclooxygenase inhibitors. The H4/18 Mab immunoprecipitatedtwo biosynthetically labeled polypeptides with molecular weights of 100kD and 120 kD. H4/18 and F(ab')2 fragments were found to partiallyinhibit HL-60 cell (premyelocytic cell line) adhesion to activated HEC,but did not affect the adhesion of normal blood cells. In contrast, MabH18/7 did block the adhesion of normal blood PMN and therefore allowedthe designation of ELAM-1 (Bevilacqua et al., Proc. Natl. Acad. Sci.(USA) 84:9238-9242 (1987).

Induction of expression of ELAM-1 on endothelial cells at the site ofinflammation has been detected at the site of inflammation using the MabH4/18 in microvascular endothelium of human skin. Cotran, R. S., et al.,J. Exp. Med. 164:661-666 (1986).

The present invention relates to cloned ELAM-1 genes. cDNA encodingELAM-1 may be obtained by stimulating cultured human endothelium cells,isolation of the mRNA, followed by preparation of cDNA by, for example,use of reverse transcriptase according to methods known in the art.Alternatively, episomal DNA may be recovered. The cDNA or episomal DNAis then shotgun cloned, and the resulting clones screened with anantibody which recognizes ELAM-1. Alternatively, the clones may bescreened with a complementary labeled probe.

Antibodies which recognize ELAM-1 may be prepared by immunizing micewith cultured human endothelial cells (HEC) which have been activatedby, for example, the monokine interleukin 1 (IL 1). Fusion of thesplenocytes with myeloma cells and subsequent screening of thehybridomas with IL 1-treated and control HEC allows selection ofmonoclonal antibodies specific for ELAM-1. See Bevilacqua, M. P., etal., 4th Int'l Symp. Biol. Vasc. Endothelial Cell:13 (Abstr) (1986);Pober, J. S., et al., J. Immunol. 136:1680-1687 (1986); Bevilacqua, M.P., et al., in: Leukocyte Immigration and Its Sequelae, Satellite Symp.(Henry Z. Movat, ed.), pp. 79-93, published by S. Karger, Basil,Switzerland (1987). Bevilacqua et al., Proc. Natl. Acad. Sci. (USA)84:9238-9242 (1987). Particularly useful monoclonal antibodies for thispurpose include H 4/18 and H 18/7.

Antibodies to ELAM-1 may also be prepared by immunizing mice withrecombinant ELAM-1, or ELAM-1 fragments. By "ELAM-1 fragment" isintended any polypeptide subset of the molecule. Particularly preferredELAM-1 fragments include leukocyte-binding fragments andcomplement-binding fragments. The leukocyte-binding orcomplement-binding fragments may be obtained by cutting the ELAM-1 genewith various restriction enzymes or exonucleases, cloning the resultingfragments, and screening for leukocyte or complement binding activityaccording to methods known in the art. Alternatively, it is possible toanalyze the ELAM-1 structure by a suitable computer program, identifythe antigenic regions, and prepare ELAM-1 fragments by methods ofprotein synthesis known to those of ordinary skill in the art.Therefore, the invention also relates to antibodies having definedspecificity obtained by immunization of an animal with an ELAM-1fragment.

The joining of various DNA fragments, is performed in accordance withconventional techniques, employing blunt-ended or staggered-endedtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate, alkaliand phosphatase treatment to avoid undesirable joining, and ligationwith appropriate ligases. The genetic construct may optionally encode aleader sequence to allow efficient expression of the recombinantprotein.

To express the recombinant ELAM-1, transcriptional and translationalsignals recognized by an appropriate host element are necessary.Mammalian cells provide post translational modification to recombinantprotein molecules which provide for correct folding and glycosylation ofappropriate sites. Mammalian cells which may be used in the practice ofthe invention include COS cells, Chinese hamster ovary (CHO) cells,leukocytes, myeloma cells or other transformed or oncogenic lymphocytes,e.g., EBV-transformed cells, cells of fibroblast origin such as VERO orcells of lymphoid origin, such as the hybridoma SP2/0-AG14 or themyeloma P3x63Sgh, and their derivatives. Other hosts include BHK cellsand hepatoma cells.

In general, vectors containing replicon and control sequences which arederived from species compatible with a host cell are used in connectionwith the host. The vector ordinarily carries a replicon site, as well asspecific genes which are capable of providing phenotypic selection intransformed cells. The expression of the gene-encoding ELAM-1 can alsobe placed under control with other regulatory sequences which may behomologous to the cell line in its untransformed state. For example,lactose-dependent E. coli chromosomal DNA comprises a lactose or lacoperon which mediates lactose utilization by elaborating the enzymebeta-galactosidase. The lac control elements may be obtained frombacterial phage lambda plac5, which is infective for E. coli. The lacpromoter-operator system can be induced by IPTG.

Other promoters/operator systems or portions thereof can be employed aswell. For example, colicin E1, galactose, alkaline phosphatase,tryptophan, xylose, and the like can be used.

For mammalian hosts, several possible vector systems are available forexpression. One class of vectors utilizes DNA elements which are derivedfrom animal viruses such as bovine papilloma virus, polyoma virus,adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV orMOMLV), or SV40 virus. Cells which have stably integrated the DNA intotheir chromosomes may be selected by introducing one or more markerswhich allow selection of transfected host cells. The marker may providefor prototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals such as copper or the like. The selectablemarker gene can be either directly linked to the DNA sequences to beexpressed, or introduced into the same cell by cotransformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionalpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements includes those described by Okayama,H., Mol. Cel. Biol., 3:280 (1983) and others.

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced to anappropriate host. Various techniques may be employed, such as protoplastfusion, calcium phosphate precipitation, electroporation or otherconventional techniques. After transfection, the cells are grown inmedia and screened for the appropriate activity using, for example, theabove-described antibodies. Expression of the gene(s) results inproduction of the ELAM-1.

The transformed cells may be grown in appropriate nutrient medium inculture flasks or injected into a synergistic host, e.g., mouse or arat, or immunodeficient host or host site, e.g., nude mouse or hamsterpouch. In particular, the cells may be introduced into the abdominalcavity of an animal to allow production of ascites fluid which containsELAM-1, or fragment thereof. Alternatively, the cells may be injectedsubcutaneously and ELAM-1 is harvested from the blood of the host. Thecells may be used in the same manner as hybridoma cells.

The expressed ELAM-1, or fragment thereof, may be isolated fromfermentation media or cell culture and purified in accordance withconventional conditions, such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis or the like.

For example, ELAM-1 may be purified by passing a solution containing thesurface protein through a column containing an immobilized antibodywhich is specific for ELAM-1, for example, the above-describedantibodies H18/7 and H4/18. The desired protein may then be eluted byraising the pH of the eluant.

ELAM-1, or fragments thereof, may be administered to a patient to treatinflammation. A soluble form of ELAM-1 may block leukocyte adhesion toendothelium at sites of inflammation. In addition, the primary structureof ELAM-1 derived from amino acid sequence analysis suggests that ELAM-1binds proteins of the complement system. Therefore, administration ofELAM-1, or the complement or leukocyte-binding fragment thereof, wouldregulate complement activation and inhibit its pathophysiologicalconsequences.

It is also possible to block inflammation by administering to a patienta monoclonal antibody directed toward ELAM-1. Such monoclonal antibodiesinclude the above-described antibodies H18/7 and H4/18. The monoclonalantibodies may also comprise antibody fragments, for example, F(ab')2fragments which minimize immunological reaction due to the Fc portion ofthe immunoglobin.

Administration of ELAM-1, or fragment thereof, may also be used for theprevention of post-reperfusion injury. When treating arterialthrombosis, induction of reperfusion by clot lysing agents such astissue plasminogen activator (t-PA) is often associated with tissuedamage. Such tissue damage is thought to be mediated, at least in partby polymorphonuclear leukocytes (PMN). Therefore, administration ofELAM-1, or a fragment thereof, preferably, a leukocyte-binding fragment,would block PMN-endothelial interactions, and thereby diminish orprevent post-reperfusion injury.

Alternatively, it is also possible to administer an antibody to apatient directed toward ELAM-1 to block PMN-endothelial interactions,and thereby prevent or treat inflammation. Suitable antibodies for thispurpose include H18/7 and H4/18, or an antibody fragment thereof.

The invention also relates to a method of detecting ELAM-1 expression ina patient, comprising

administering to a patient a pharmaceutical composition comprising adetectable amount of a labeled ELAM-1 specific antibody, or antibodyfragment, and a pharmaceutically acceptable carrier; and

detecting the label.

Preferably, the labels which can be detected within the body of apatient include, but are not limited to radiopaque labels, radiolabelsor paramagnetic isotopes. This method allows the detection andlocalization of ELAM-1 expression in a patient which is associated withvarious disease states.

After the ELAM-1 specific antibody, or antibody fragment, has beenadministered and sufficient time has passed for the antibody to localizeat the site of ELAM-1 expression and unbound antibody has been permittedto clear from healthy tissue in the patient, the label may be detected.Where the label is a radionuclide, it must be of the type of decay whichis detectable for a given type of instrument. Further, the radionuclidefor in vivo diagnosis should have a half-life long enough so that it isstill detectable at the time of maximum uptake, but short enough thatafter diagnosis unwanted radiation does not remain in the patient.Coupling of the radionuclides to the antibody or antibody fragment isknown in the art and is often accomplished either directly or indirectlyusing an intermediary functional group. Examples of radioisotopes thatcan be used for in vivo diagnosis are ⁹⁹ Tc, ¹²³ I, ¹³¹ I, ¹¹¹ In, ⁹⁷Ru, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr, and ²⁰¹ Tl.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. Examples of elements thatare particularly useful for use in Magnetic Resonance Energy techniquesinclude ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe.

It is also possible to use antibodies directed to ELAM-1 to detect thepresence of ELAM-1 in biological samples such as blood, serum,cerebrospinal fluid, etc., as an indication of expression of ELAM-1 inassociation with disease various processes.

The detection and quantitation of antigenic substances and biologicalsamples frequently utilize immunoassay techniques. These techniques arebased upon the formation of a detectable complex between the antigenicsubstance being assayed, e.g., ELAM-1, and an antibody or antibodies inwhich one or the other member of the complex may be detectably labeled.In the present invention, the ELAM-1 specific antibody may be labeledwith any conventional label.

Thus, in this aspect of the invention, a biological sample may betreated with nitrocellulose, or other solid support which is capable ofimmobilizing cells, cell particles or soluble protein. The support maythen be washed with suitable buffers followed by treatment with thedetectably labeled ELAM-1 specific antibody. The solid phase support maythen be washed with the buffer a second time to remove unbound antibody.The amount of bound label on the antibody may then be detected byconventional means.

In carrying out the assay of the present invention on a samplecontaining ELAM-1 or cells expressing ELAM-1 on their surface, theprocess comprises:

(a) contacting a sample suspected containing ELAM-1 or cell expressingELAM-1 on its surface with a solid support to effect immobilization ofELAM-1, or cell which expresses ELAM-1 on its surface;

(b) contacting said solid support with a detectably labeled ELAM-1specific antibody;

(c) incubating said detectably labeled ELAM-1 specific antibody withsaid support for a time sufficient to allow the ELAM-1 specific antibodyto bind to the immobilized ELAM-1 or cell which expresses ELAM-1 on itssurface;

(d) separating the solid phase support from the incubation mixtureobtained in step (c); and

(e) detecting the bound label and thereby detecting and quantifyingELAM-1 or cell expressing ELAM-1 on its surface.

Alternatively, labeled ELAM-1 specific antibody/ELAM-1 complex in asample may be separated from a reaction mixture by contacting thecomplex with an immobilized antibody or protein which is specific for animmunoglobulin, e.g., protein A, protein G, anti-IgM or anti-IgGantibodies. Such anti-immunoglobulin antibodies may be monoclonal orpolyclonal. The solid support may then be washed with a suitable bufferto give an immobilized ELAM-1/labeled ELAM-1 specific antibody complex.The label on the fusion protein may then be detected to give a measureof endogenous ELAM-1 and, thereby, the extent of stimulated endothelialcells.

This aspect of the invention relates to a method for detecting ELAM-1 orleukocyte-binding fragment thereof in a sample comprising

(a) contacting a sample suspected of containing ELAM-1 with an ELAM-1specific antibody or fragment thereof which binds to ELAM-1;

(b) detecting whether a complex is formed.

The invention also relates to a method of detecting ELAM-1 in a sample,further comprising

(c) contacting the mixture obtained in step (a) with an Fc bindingmolecule, such as an antibody, protein A, or protein G, which isimmobilized on a solid phase support and is specific for the ELAM-1specific antibody to give a ELAM-1/ELAM-1 specific antibody-immobilizedantibody complex;

(d) washing the solid phase support obtained in step (c) to removeunbound ELAM-1/ELAM-1 specific antibody complex;

(e) and detecting the label on the ELAM-1 specific antibody.

Of course, the specific concentrations of detectably labeled antibodyand ELAM-1, the temperature and time of incubation, as well as otherassay conditions may be varied, depending on various factors includingthe concentation of ELAM-1 in the sample, the nature of the sample, andthe like. Those skilled in the art will be able to determine operativeand optimal assay conditions for each determination by employing routineexperimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which the ELAM-1 specific antibody can be detectablylabeled is by linking the same to an enzyme. This enzyme, in turn, whenlater exposed to its substrate, will react with the substrate in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used to detectably label the ELAM-1 specific antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholineesterase.

The ELAM-1 specific antibody may also be labeled with a radioactiveisotope which can be determined by such means as the use of a gammacounter or a scintillation counter or by audioradiography. Isotopeswhich are particularly useful for the purpose of the present inventionare: ³ H, ¹²⁵ I, ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹Fe and ⁷⁵ Se.

It is also possible to label the ELAM-1 specific antibody with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to the fluorescence of the dye. Among the most commonly usedfluorescent labelling compounds are fluorescein isothiocyanate,rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The ELAM-1 specific antibody can also be detectably labeled usingfluorescence emitting metals such as ¹⁵² Eu, or others of the lanthanideseries. These metals can be attached to the ELAM-1 specific antibodyusing such metal chelating groups as diethylenetriaminepentaacetic acid(DTPA) or ethylenedtaminetetraacetic acid (EDTA).

The ELAM-1 specific antibody also can be detectably labeled by couplingit to a chemiluminescent compound. The presence of thechemiluminescent-tagged ELAM-1 specific antibody is then determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the ELAM-1specific antibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

Detection of the ELAM-1 specific antibody may be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorimetric methods which employ asubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

ELAM-1, or leukocyte-binding fraction thereof, may also be administeredto a patient to prevent adhesion of leukocyte tumor cells ornon-leukocyte tumor cells to endothelial tissue. Thus, administration ofELAM-1, or leukocyte-binding fragment thereof, would prevent metastaticspread of tumor cells. Alternatively, antibodies directed to ELAM-1, orantibody fragments, may be administered to block the adhesion of tumorcells to endothelial tissue, thereby inhibiting metastatic spread.

In addition, ELAM-1, or a leukocyte-binding fragment thereof, may becoupled to a chemotherapeutic drug which could bind to tumor cellsexpressing receptors for ELAM-1, to kill the tumor cell. Thus, theinvention also relates to a method for treating, for example, leukemiacomprising administering to a patient ELAM-1, or a leukocyte-bindingfragment thereof, which is coupled to an anti-tumor agent. Suchantitumor agents may include, for example, bacterial toxins such asdiphtheria toxin or ricin, a toxic lectin.

Thus, the invention also relates to fusion proteins comprising ELAM-1,or leukocyte-binding fragment thereof, coupled to a second proteincomprising diphtheria toxin or ricin. Methods for producing fusionproteins comprising fragment A of diphtheria toxin by recombinant meansare taught in U.S. Pat. No. 4,675,382 (1987). Diphtheria toxin containstwo polypeptide chains. The B chain binds the toxin to a receptor on acell surface. The A chain actually enters the cytoplasm and inhibitsprotein synthesis by inactivating elongation factor 2, the factor thattranslocates ribosomes along mRNA concomitant with hydrolysis of ATP.See Darnell, J., et al., in Molecular Cell Biology, Scientific AmericanBooks, Inc., page 662 (1986). In a preferred embodiment, the fusionprotein of the invention comprises ELAM-1, or a leukocyte-bindingfragment thereof, and the A chain of diphtheria toxin.

ELAM-1 is also useful for the treatment of microbial infections. Avariety of microbes (bacteria, rickettsia, borrelia, various parasites,and viruses) bind to vascular endothelium in some cases, possibly, by anELAM-1 dependent mechanism. Thus, ELAM-1, or a leukocyte-bindingfragment thereof, may be administered to a patient to prevent thebinding of microbes which recognize ELAM-1 as a binding target moleculeand thereby, treat microbial infections.

Moreover, it is also possible to administer an antibody or antibodyfragment directed to ELAM-1, to block the microbe receptor onendothelial tissue. Thus, the invention also relates to the treatment ofmicrobial infections comprising administering an antibody, or antibodyfragment, directed to ELAM-1 to a patient.

The invention also relates to a treatment for microbial infectionscomprising administering a pharmaceutical composition comprising ELAM-1,or a fragment thereof, which is coupled to an antimicrobial agent. Sucha conjugate specifically delivers and localizes the antimicrobial agentat the site of vascular infection.

Such antimicrobial agents include ricin or diphtheria toxin, asdescribed above, or may comprise halogenated amino acids which act assuicide enzyme inhibitors. See Soper, T. S., et al., J. Biol. Chem.252:3170-3175 (1977) (β-chloroalanine); Kollonitsch, J., et al., J.Amer. Chem. Soc. 98:5591-5593 (1976) (3-fluoroalanine); Wang, E., etal., Biochemistry 17:1313-1321 (1978) (β,β-difluoroalanine andβ,β,β-trifluoroalanine).

The invention also relates to a method of treating vasculitis byadministering ELAM-1, or a fragment thereof, to a patient. Recentevidence suggests that tissue damage in certain forms of vasculitisinvolves induced endothelial surface proteins. Thus, ELAM-1 may be atarget for antibody-mediated or cell-mediated damage of the vessel wall.

ELAM-1, or a structurally homologous molecule, may be involved inmediating cell-cell interactions in embryogenesis/organ development; inneoplasia (with possible expression by tumor cells); in wound/tissueregeneration; and in related processes. Therefore, administration ofELAM-1, or fragments thereof, is useful as a therapeutic interventionand/or a diagnostic tool for investigation of these processes.

The ELAM-1 molecule, or fragment thereof, or antibodies and antibodyfragments directed to ELAM-1, can be formulated into pharmaceuticallyuseful compositions according to known methods, such as by admixturewith a pharmaceutically acceptable carrier vehicle. Suitable vehiclesand their formulation are described, for example, in Remington'sPharmaceutical Sciences (16th ed.), Osol, A. (ed.), Mack Publishers,Easton (1980). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain a therapeutically effective amount of the ELAM-1, fragmentof ELAM-1, or antibody or antibody fragment directed to ELAM-1, eitheralone or with a suitable amount of carrier vehicle.

When used for the treatment of inflammation, post-reperfusion injury,leukemia, lymphoma, microbial/parasitic infection, vasculitis, orinhibition of the metastatic spread of tumor cells, the pharmaceuticalcomposition may comprise from 1 pg/kg to 10 mg/kg patient of ELAM-1,fragment of ELAM-1, or fusion protein of the invention, although higheror lower doses are possible. When used for the purpose of treatinginflammation, microbial/parasitic infection, post-reperfusion injury,leukemia, lymphoma, vasculitis or inhibition of the metastatic spread oftumor cells, the pharmaceutical composition may comprise from 1 pg/kg to10 mg/kg of ELAM-specific antibody or antibody fragment thereof,although higher or lower doses are possible.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the ELAM-1, ELAM-1 fragment orantibody or antibody fragment directed to ELAM-1 of the invention.Controlled delivery may be achieved by selecting appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers). The rate of drug release may also be controlled by alteringthe concentration of such macromolecules. Another possible method forcontrolling the duration of action comprises incorporating thetherapeutic agents into particles of a polymeric substance such aspolyesters, polyamino acids, hydrogels, polylactide/glycolidecopolymers, or ethylenevinylacetate copolymers. Alternatively, it ispossible to entrap the therapeutic agents in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, by the use of hydroxymethylcellulose orgelatin-microcapsules or poly(methylmethacrolate) microcapsules,respectively, or in a colloid drug delivery system, for example,liposomes, albumin microspheres, microemulsions, nanoparticles,nanocapsules, or in macroemulsions. Such teachings are disclosed inRemington's Pharmaceutical Sciences (1980).

ELAM-1, fragment of ELAM-1, or antibody or antibody fragment directed toELAM-1 may be provided to a patient by means well known in the art. Suchmeans of introduction include oral means, intranasal means, subcutaneousmeans, intramuscular means, intravenous means, intra-arterial means, orparenteral means.

Having now generally described this invention, the same will be betterunderstood with reference to certain specific examples which are hereinincluded for purposes of illustration only, and are not intended to belimiting of the invention, unless specified.

EXAMPLES Example 1 Molecular Cloning of ELAM-1

A cDNA library was constructed using RNA obtained from cultured HEC thathad been treated for 2 hr with rIL-lβ (5 U/ml). The library wastransfected into COS cells and Mabs H18/7 and H4/18 were used to isolatecells expressing ELAM-1 epitopes. Episomal DNA was recovered, expandedin bacteria (mc 1061p3), and subjected to two more rounds ofexpression-panning (Seed, B., and Aruffo, A., Proc. Natl. Acad. Sci.(USA) 84:3365-3369 (1987); Aruffo, A., and Seed, B., Proc. Natl. Acad.Sci. (USA) 84:8573-8577 (1987)) to obtain a cDNA clone designatedpELAM-1. COS cells transfected with pELAM-1 expresses a molecule(s)reactive with both Mab H18/7 and H4/18, but not with control Mabs, asdetected by indirect immunofluorescence and immunoperoxidase.

Mab H18/7 immunoprecipitated two polypeptides of Mr 115,000 and 97,000from biosynthetically labeled, rIL-1 stimulated HEC but not from controlHEC. Two proteins of similar mass (Mr 107,000 and 97,000) were obtainedfrom biosynthetically labeled, pELAM-1 transfected COS cells. The lowermolecular mass of the COS cell product is consistent with previousobservations (Seed, B., and Aruffo, A., Proc. Natl. Acad. Sci. (USA)84:3365-3369 (1987); Aruffo, A., and Seed, B., Proc. Natl. Acad. Sci.(USA) 84:8573-8577 (1987)), and may reflect altered glycosylation.Treatment of both the COS cell and HEC derived immunoprecipitates withN-glycosidase F (N-Glycanase, Genzyme Inc., Boston, Mass.) to removeN-linked carbohydrates, yielded a single band by electrophoresiscorresponding to a polypeptide(s) of Mr 77,000. These data suggest thatthe polypeptide backbone of recombinant, COS cell-derived ELAM-1 isequivalent to that of natural, endothelial cell-derived ELAM-1. Inaddition, it is consistent with the hypothesis that the two speciesimmunoprecipitated from biosynthetically labeled cells contain the samepolypeptide backbone and differ in post-translational modification. Thishypothesis is further supported by pulse-chase biosynthetic labelingexperiments using rIL-1β stimulated HEC and pELAM-1 transfected COScells. In both cases, label is first seen associated with the lowermolecular mass protein (Mr 97,000), followed by an apparent "chasing" ofthe label into the upper molecular mass form. Taken together, these datastrongly suggest that the two species immunoprecipitated withanti-ELAM-1 Mabs are related as precursor and product.

Example 2 Primary Structure and Homologies

As represented in FIG. 1, pELAM-1 was found to be 3.85 kb with a short5' untranslated region, a 1,830 base open reading frame, and arelatively long (1.9 kb) 3' untranslated region. The nucleotide sequenceof the cDNA encoding ELAM-1 and the 3' untranslated region is shownbelow:

    __________________________________________________________________________      1                                                                              CCTGAGACAG                                                                             AGGCAGCAGT                                                                              GATACCCACC                                                                              TGAGAGATCC                                                                              TGTGTTTGAA                           51                                                                              CAACTGCTTC                                                                             CCAAAACGGA                                                                              AAGTATTTCA                                                                              ACCCTAAACC                                                                              TTTGGGTGAA                           101                                                                             AAGAACTCTT                                                                             GAAGTCATGA                                                                              TTGCTTCACA                                                                              GTTTCTCTCA                                                                              GCTCTCACTT                           151                                                                             TGGTGCTTCT                                                                             CATTAAAGAG                                                                              AGTGCAGCCT                                                                              GGTCTTACAA                                                                              CACCTCCACG                           201                                                                             GAAGCTATGA                                                                             CTTATGATGA                                                                              GGCCAGTGCT                                                                              TATTGTCAGC                                                                              AAAGGTACAC                           251                                                                             ACACCTGGTT                                                                             GCAATTCAAA                                                                              ACAAAGAAGA                                                                              GATTGACTAC                                                                              CTAAACTCCA                           301                                                                             TATTGAGCTA                                                                             TTCACCAAGT                                                                              TATTACTGGA                                                                              TTGGAATCAG                                                                              AAAAGTCAAC                           351                                                                             AATGTGTGGG                                                                             TCTGGGTAGG                                                                              AACCCAGAAA                                                                              CCTCTGACAG                                                                              AAGAAGCCAA                           401                                                                             GAACTGGGCT                                                                             CCAGGTGAAC                                                                              CCAACAATAG                                                                              GCAAAAAGAT                                                                              GAGGACTGCG                           451                                                                             TGGAGATCTA                                                                             CATCAAGAGA                                                                              GAAAAAGATG                                                                              TGGGCATGTG                                                                              GAATGATGAG                           501                                                                             AGGTGCACCA                                                                             AGAAGAAGCT                                                                              TGCCCTATGC                                                                              TACACAGCTG                                                                              CCTGTACCAA                           551                                                                             TACATCCTGC                                                                             AGTGGCCACG                                                                              GTGAATGTGT                                                                              AGAGACCATC                                                                              AATAATTACA                           601                                                                             CTTGCAAGTG                                                                             TGACCCTGGC                                                                              TTCAGTGGAC                                                                              TCAAGTGTGA                                                                              GCAAATTGTG                           651                                                                             AACTGTACAG                                                                             CCCTGGAATC                                                                              CCCTGAGCAT                                                                              GGAAGCCTGG                                                                              TTTGCAGTCA                           701                                                                             CCCACTGGGA                                                                             AACTTCAGCT                                                                              ACAATTCTTC                                                                              CTGCTCTATC                                                                              AGCTGTGATA                           751                                                                             GGGGTTACCT                                                                             GCCAAGCAGC                                                                              ATGGAGACCA                                                                              TGCAGTGTAT                                                                              GTCCTCTGGA                           801                                                                             GAATGGAGTG                                                                             CTCCTATTCC                                                                              AGCCTGCAAT                                                                              GTGGTTGAGT                                                                              GTGATGCTGT                           851                                                                             GACAAATCCA                                                                             GCCAATGGGT                                                                              TCGTGGAATG                                                                              TTTCCAAAAC                                                                              CCTGGAAGCT                           901                                                                             TCCCATGGAA                                                                             CACAACCTGT                                                                              ACATTTGACT                                                                              GTGAAGAAGG                                                                              ATTTGAACTA                           951                                                                             ATGGGAGCCC                                                                             AGAGCCTTCA                                                                              GTGTACCTCA                                                                              TCTGGGAATT                                                                              GGGACAACGA                          1001                                                                             GAAGCCAACG                                                                             TGTAAAGCTC                                                                              TGACATGCAG                                                                              GGCCGTCCGC                                                                              CAGCCTCAGA                          1051                                                                             ATGGCTCTGT                                                                             CAGGTGCAGC                                                                              CATTCCCCTG                                                                              CTGGAGAGTT                                                                              CACCTTCAAA                          1101                                                                             TCATCCTGCA                                                                             ACTTCACCTG                                                                              TGAGGAAGGC                                                                              TTCATGTTGC                                                                              AGGGACCAGC                          1151                                                                             CCAGGTTGAA                                                                             TGCACCACTC                                                                              AAGGGCAGTG                                                                              GACACAGCAA                                                                              ATCCCAGTTT                          1201                                                                             GTGAAGCTTT                                                                             CCAGTGCACA                                                                              GCCTTGTCCA                                                                              ACCCCCAGCG                                                                              AGGCTACATG                          1251                                                                             AATTGTCTTC                                                                             CTAGTGCTTC                                                                              TGGCAGTTTC                                                                              CGTTATGGGT                                                                              CCAGCTGTGA                          1301                                                                             GTTCTCCTGT                                                                             GAGCAGGGTT                                                                              TTGTGTTGAA                                                                              GGGATCCAAA                                                                              AGGCTCCAAT                          1351                                                                             GTGGCCCCAC                                                                             AGGGGAGTGG                                                                              GACAACGAGA                                                                              AGCCCACATG                                                                              TGAAGCTGTG                          1401                                                                             AGATGCGATG                                                                             CTGTCCACCA                                                                              GCCCCCGAAG                                                                              GGTTTGGTGA                                                                              GGTGTGCTCA                          1451                                                                             TTCCCCTATT                                                                             GCAGAATTCA                                                                              CCTACAAGTC                                                                              CTCTTGTGCC                                                                              TTCAGCTGTG                          1501                                                                             AGGAGGGATT                                                                             TGAATTATAT                                                                              GGATCAACTC                                                                              AACTTGAGTG                                                                              CACATCTCAG                          1551                                                                             GGACAATCGA                                                                             CAGAAGAGGT                                                                              TCCTTCCTCC                                                                              CAAGTGGTAA                                                                              AATGTTCAAG                          1601                                                                             CCTGGCAGTT                                                                             CCGGGAAAGA                                                                              TCAACATGAG                                                                              CTGCAGTGGG                                                                              GAGCCCGTGT                          1651                                                                             TTGGCACTGT                                                                             GTGCAAGTTC                                                                              GCCTGTCCTG                                                                              AAGGATGGAC                                                                              GCTCAATGGC                          1701                                                                             TCTGCAGCTC                                                                             GGACATGTGG                                                                              AGCCACAGGA                                                                              CACTGGTCTG                                                                              GCCTGCTACC                          1751                                                                             TACCTGTGAA                                                                             GCTCCCACTG                                                                              AGTCCAACAT                                                                              TCCCTTGGTA                                                                              GCTGGACTTT                          1801                                                                             CTGCTGCTGG                                                                             ACTCTCCCTC                                                                              CTGACATTAG                                                                              CACCATTTCT                                                                              CCTCTGGCTT                          1851                                                                             CGGAAATGCT                                                                             TACGGAAAGC                                                                              AAAGAAATTT                                                                              GTTCCTGCCA                                                                              GCAGCTGCCA                          1901                                                                             AAGCCTTGAA                                                                             TCAGACGGAA                                                                              GCTACCAAAA                                                                              GCCTTCTTAC                                                                              ATCCTTTAAG                          1951                                                                             TTCAAAAGAA                                                                             TCAGAAACAG                                                                              GTGCATCTGG                                                                              GGAACTAGAG                                                                              GGATACACTG                          2001                                                                             AAGTTAACAG                                                                             AGACAGATAA                                                                              CTCTCCTCGG                                                                              GTCTCTGGCC                                                                              CTTCTTGCCT                          2051                                                                             ACTATGCCAG                                                                             ATGCCTTTAT                                                                              GGCTGAAACC                                                                              GCAACACCCA                                                                              TCACCACTTC                          2101                                                                             AATAGATCAA                                                                             AGTCCAGCAG                                                                              GCAAGGACGG                                                                              CCTTCAACTG                                                                              AAAAGACTCA                          2151                                                                             GTGTTCCCTT                                                                             TCCTACTCTC                                                                              AGGATCAAGA                                                                              AAGTGTTGGC                                                                              TAATGAAGGG                          2201                                                                             AAACCATATT                                                                             TTCTTCCAAG                                                                              CAAAGGTGAA                                                                              GAGACCAAGA                                                                              CTCTGAAATC                          2251                                                                             TCAGAATTCC                                                                             TTTTCTAACT                                                                              CTCCCTTGCT                                                                              CGCTGTAAAA                                                                              TCTTGGCACA                          2301                                                                             GAAACACAAT                                                                             ATTTTGTGGC                                                                              TTTCTTTCTT                                                                              TTGCCCTTCA                                                                              CAGTGTTTCG                          2351                                                                             ACAGCTGATT                                                                             ACACAGTTGC                                                                              TGTCATAAGA                                                                              ATGAATAATA                                                                              ATTATCCAGA                          2401                                                                             GTTTAGAGGA                                                                             AAAAAATGAC                                                                              TAAAAATATT                                                                              ATAACTTAAA                                                                              AAAATGACAG                          2451                                                                             ATGTTGAATG                                                                             CCCACAGGCA                                                                              AATGCATGGA                                                                              GGGTRGTTAA                                                                              TGGTGCAAAT                          2501                                                                             CCTACTGAAT                                                                             GCTCTGTGCG                                                                              AGGGTTACTA                                                                              TGCACAATTT                                                                              AATCACTTTC                          2551                                                                             ATCCCTATCG                                                                             GATTCAGTGC                                                                              TTCTTAAACA                                                                              GTTCTTAAGG                                                                              ATTGTGATAT                          2601                                                                             TTTTACTTGC                                                                             ATTGAATATA                                                                              TRATAATCTT                                                                              CCATACTTCT                                                                              TCATTCAATA                          2651                                                                             CAAGTGTGGT                                                                             AGGGACTTAA                                                                              AAAACTTGTA                                                                              AATGCTGTCA                                                                              ACTATGATAT                          2701                                                                             GGTAAAAGTT                                                                             ACTTATTCTA                                                                              GATTACCCCC                                                                              TCATRGTTTA                                                                              TRAACAAATT                          2751                                                                             ATGTTACATC                                                                             TGTTTTAAAT                                                                              TTATTTCAAA                                                                              AAGGGAAACT                                                                              ATTGTCCCCT                          2801                                                                             AGCAAGGCAT                                                                             GATGTTAACC                                                                              AGAATAAACT                                                                              TCTGAGTGTT                                                                              TTTACTACAG                          2851                                                                             TTGTTTTTTG                                                                             AAAACATGGT                                                                              AGAATTGGAG                                                                              AGTAAAAACT                                                                              GAATGGAAGG                          2901                                                                             TTTGTATATT                                                                             GTCAGATATT                                                                              TTTTCAGAAA                                                                              TATGTGGTTT                                                                              CCACGATGAA                          2951                                                                             AAACTTCCAT                                                                             GAGGCCAAAC                                                                              GTTTTGAACT                                                                              AATAAAAGCA                                                                              TAAATGCAAA                          3001                                                                             CACACAAAGG                                                                             TATAATTTTA                                                                              TGAATGTCTT                                                                              TGTTGGAAAA                                                                              GAATACAGAA                          3061                                                                             AGATGGATGT                                                                             GCTTTGCATT                                                                              CCTACAAAGA                                                                              TGTTTGTCAG                                                                              ATGTGATATG                          3101                                                                             TAAACATAAT                                                                             TCTTGTATAT                                                                              TATGGAAGAT                                                                              TTTAAATTCA                                                                              CAATAGAAAC                          3151                                                                             TCACCATGTA                                                                             AAAGAGTCAT                                                                              CTGGTAGATT                                                                              TTTAACGAAT                                                                              GAAGATGTCT                          3201                                                                             AATAGTTATT                                                                             CCCTATTTGT                                                                              TTTCTTCTGT                                                                              ATGTTAGGGT                                                                              GCTCTGGAAG                          3251                                                                             AGAGGAATGC                                                                             CTGTGTGAGC                                                                              AAGCATTTAT                                                                              GTTTATTTAT                                                                              AAGCAGATTT                          3301                                                                             AACAATTCCA                                                                             AAGGAATCTC                                                                              CAGTTTTCAG                                                                              TTGATCACTG                                                                              GCAATGAAAA                          3351                                                                             ATTCTCAGTC                                                                             AGTAATRGCC                                                                              AAAGCTGCTC                                                                              TAGCCTTGAG                                                                              GAGTGTGAGA                          3401                                                                             ATCAAAACTC                                                                             TCCTACACTT                                                                              CCATTAACTT                                                                              AGCATGTGTT                                                                              GAAAAAAAAA                          3451                                                                             GTTTCAGAGA                                                                             AGTTCTGGCT                                                                              GAACACTGGC                                                                              AACGACAAAG                                                                              CCAACAGTCA                          3501                                                                             AAACAGAGAT                                                                             GTGATAAGGA                                                                              TCAGAACAGC                                                                              AGACGTTCTT                                                                              TTAAAGGGGC                          3551                                                                             AGAAAAACTC                                                                             TGGGAAATAA                                                                              GAGAGAACAA                                                                              CTACTGTGAT                                                                              CAGGCTATGT                          3601                                                                             ATGGAATACA                                                                             GTGTTATTTT                                                                              CTTTGAAATT                                                                              GTTTAAGTGT                                                                              TGTAAATATT                          3651                                                                             TATGTAAACT                                                                             GCATTAGAAA                                                                              TTAGCTGTGT                                                                              GAAATACCAG                                                                              TGTGGTTTGT                          3701                                                                             GTTTGAGTTT                                                                             TATTGAGAAT                                                                              TTTAAATTAT                                                                              AACTTAAAAT                                                                              ATTTTATAAT                          3751                                                                             TTTTAAAGTA                                                                             TATATTTATT                                                                              TAAGCTTATG                                                                              TCAGACCTAT                                                                              TTGACATAAC                          3801                                                                             ACTATAAAGG                                                                             TTGACAATAA                                                                              ATGTGCTTAT                                                                              GTTTAAAAAA                                                                              AAAAAAAAAA                          3851                                                                             AAAA                                                                       __________________________________________________________________________

The nucleotide sequence of the cDNA encoding ELAM-1 is shown below:

    __________________________________________________________________________                ATGA      TTGCTTCACA                                                                              GTTTCTCTCA                                                                              GCTCTCACTT                           151                                                                             TGGTGCTTCT                                                                             CATTAAAGAC                                                                              AGTGGAGCCT                                                                              GGTCTTACAA                                                                              CACCTCCACG                           201                                                                             GAAGCTATGA                                                                             CTTATGATGA                                                                              GGCCAGTGCT                                                                              TATTGTCAGC                                                                              AAAGGTACAC                           251                                                                             ACACCTGGTT                                                                             GCAATTCAAA                                                                              ACAAAGAAGA                                                                              GATTGAGTAC                                                                              CTAAACTCCA                           301                                                                             TATTGAGCTA                                                                             TTCACCAAGT                                                                              TATTACTGGA                                                                              TTGGAATCAG                                                                              AAAAGTCAAC                           351                                                                             AATGTGTGGG                                                                             TCTGGGTAGG                                                                              AACCCAGAAA                                                                              CCTCTGACAG                                                                              AAGAAGCCAA                           401                                                                             GAACTGGGCT                                                                             CCAGGTGAAC                                                                              CCAACAATAG                                                                              GCAAAAAGAT                                                                              GAGGACTGCG                           451                                                                             TGGAGATCTA                                                                             CATCAAGAGA                                                                              GAAAAAGATG                                                                              TGGGCATGTG                                                                              GAATGATGAG                           501                                                                             AGGTGCAGCA                                                                             AGAAGAAGCT                                                                              TGCCCTATGC                                                                              TACACAGCTG                                                                              CCTGTACCAA                           551                                                                             TACATCCTGC                                                                             AGTGGCCACG                                                                              GTGAATGTGT                                                                              AGAGACCATC                                                                              AATAATTACA                           601                                                                             CTTGCAAGTG                                                                             TGACCCTGGC                                                                              TTCAGTGGAC                                                                              TCAAGTGTGA                                                                              GCAAATTGTG                           651                                                                             AACTGTACAG                                                                             CCCTGGAATC                                                                              CCCTGAGCAT                                                                              GGAAGCCTGG                                                                              TTTGCAGTCA                           701                                                                             CCCACTGGGA                                                                             AACTTCAGCT                                                                              ACAATTCTTC                                                                              CTGCTCTATC                                                                              AGCTGTGATA                           751                                                                             GGGGTTACCT                                                                             GCCAAGCAGC                                                                              ATGGAGACCA                                                                              TGCAGTGTAT                                                                              GTCCTCTGGA                           801                                                                             GAATGGAGTG                                                                             CTCCTATTCC                                                                              AGCCTGCAAT                                                                              GTGGTTGAGT                                                                              GTGATGCTGT                           851                                                                             GACAAATCCA                                                                             GCCAATGGGT                                                                              TCGTGGAATG                                                                              TTTCCAAAAC                                                                              CCTGGAAGCT                           901                                                                             TCCCATGGAA                                                                             CACAACCTGT                                                                              ACATTTGACT                                                                              GTGAAGAAGG                                                                              ATTTGAACTA                           951                                                                             ATGGGAGCCC                                                                             AGAGCCTTCA                                                                              GTGTACCTCA                                                                              TCTGGGAATT                                                                              GGGACAACGA                          1001                                                                             GAAGCCAACG                                                                             TGTAAAGCTG                                                                              TGACATGCAG                                                                              GGCCGTCCGC                                                                              CAGCCTCAGA                          1051                                                                             ATGGCTCTGT                                                                             GAGGTGCAGC                                                                              CATTCCCCTG                                                                              CTGGAGAGTT                                                                              CACCTTCAAA                          1101                                                                             TCATCCTGCA                                                                             ACTTCACCTG                                                                              TGAGGAAGGC                                                                              TTCATGTTGC                                                                              AGGGACCAGC                          1151                                                                             CCAGGTTGAA                                                                             TGCACCACTC                                                                              AAGGGCAGTG                                                                              GACACAGCAA                                                                              ATCCCAGTTT                          1201                                                                             GTGAAGCTTT                                                                             CCAGTGCACA                                                                              GCCTTGTCCA                                                                              ACCCCGAGCG                                                                              AGGCTACATG                          1251                                                                             AATTGTCTTC                                                                             CTAGTGCTTC                                                                              TGGCAGTTTC                                                                              CGTTATGGGT                                                                              CCAGCTGTGA                          1301                                                                             GTTCTCCTGT                                                                             GAGCAGGGTT                                                                              TTCTCTTGAA                                                                              GGGATCCAAA                                                                              AGGCTCCAAT                          1351                                                                             GTGGCCCCAC                                                                             AGGGGACTGG                                                                              GACAACGAGA                                                                              AGCCCACATC                                                                              TGAAGCTGTC                          1401                                                                             AGATGCGATC                                                                             CTGTCCACCA                                                                              GCCCCCGAAG                                                                              GCTTTGGTGA                                                                              GGTGTGCTCA                          1451                                                                             TTCCCCTATT                                                                             GGAGAATTCA                                                                              CCTACAAGTC                                                                              CTCTTGTGCC                                                                              TTCAGCTGTG                          1501                                                                             AGGAGGGATT                                                                             TGAATTATAT                                                                              GGATCAACTC                                                                              AACTTGAGTC                                                                              CACATCTCAG                          1551                                                                             GGACAATGGA                                                                             CAGAAGAGGT                                                                              TCCTTCCTGC                                                                              CAAGTGGTAA                                                                              AATGTTCAAG                          1601                                                                             CCTGGCAGTT                                                                             CCGGGAAAGA                                                                              TCAACATGAG                                                                              CTGCAGTGGG                                                                              GAGCCCGTGT                          1651                                                                             TTGGCACTGT                                                                             GTGCAAGTTC                                                                              GCCTGTCCTG                                                                              AAGGATGGAC                                                                              GCTCAATGGC                          1701                                                                             TCTGCAGCTC                                                                             GGACATGTGG                                                                              AGCCACAGGA                                                                              CACTGGTCTG                                                                              GCCTGCTACC                          1751                                                                             TACCTGTGAA                                                                             GCTCCCACTG                                                                              AGTCCAACAT                                                                              TCCCTTGGTA                                                                              GCTGGACTTT                          1801                                                                             CTGCTGCTGG                                                                             ACTCTCCCTC                                                                              CTGACATTAG                                                                              CACCATTTCT                                                                              CCTCTGGCTT                          1851                                                                             CGGAAATGCT                                                                             TACGGAAAGC                                                                              AAAGAAATTT                                                                              GTTCCTGCCA                                                                              GCAGCTGCCA                          1901                                                                             AAGCCTTGAA                                                                             TCAGACGGAA                                                                              GCTACCAAAA                                                                              GCCTTCTTAC                                                                              ATCCTTTAA                           __________________________________________________________________________

The translated amino acid sequence of ELAM-1 is shown below:

    __________________________________________________________________________                                        M I                                       A S Q F L S A L T L V L L I K E S G A  W                                      S Y N T S T E A M T Y D E A S A Y C Q  Q                                      R Y T H L V A I Q N K E E I E Y L N S  I                                      L S Y S P S Y Y W I G I R K V N N V W  V                                      W V G T Q K P L T E E A K N W A P G E  P                                      N N R Q K D E D C V E I Y I K R E K D  V                                      G M W N D E R C S K K K L A L C Y T A  A                                      C T N T S C S G H G E C V E T I N N Y  T                                      C K C D P G F S G L K C E Q I V N C T  A                                      L E S P E H G S L V C S H P L G N F S  Y                                      N S S C I S S C D R G Y L P S S M E T  M                                      Q C M S S G E W S A P I P A C N V V E  C                                      D A V T N P A N G F V E C F Q N P G S  F                                      P W N T T C T F D C E E G F E L M G A  Q                                      S L Q C T S S G N W D N E K P T C K A  V                                      T C R A V R Q P Q N G S V R C S H S P  A                                      G E F T F K S S C N F T C E E G F M L  Q                                      G P A G V E C T T Q G Q W T Q Q I P V  C                                      E A F Q C T A L S N P E R G Y M N C L  P                                      S A S G S F R Y G S S C E F S C E Q G  F                                      V L K G S K R L Q C G P T G E W D N E  K                                      P T C E A V R C D A V H Q P P K G L V  R                                      C A H S P I G E F T Y K S S C A F S C  E                                      E G F E L Y G S T Q L E C T S Q G Q W  T                                      E E V P S C Q V V K C S S L A V P G K  I                                      N M S C S G E P V F G T V C K F A C P  E                                      G W T L N G S A A R T C G A T G H W S  G                                      L L P T C E A P T E S N I P L V A G L  S                                      A A G L S L L T L A P F L L W L R K C  L                                      R K A K K F V P A S S C Q S L E S D G  S                                      Y Q K P S Y I L                                                               __________________________________________________________________________

The translated amino acid sequence of pELAM-1 suggests typical featuresof a transmembrane protein. For example, the extracellular domaincontains eleven potential N-linked glycosylation sites.

Example 3 Functional Studies

Mab 18/7 inhibits the adhesion of PMN (>50%) and HL-60 cells (>60) toactivated HEC monolayers. Bevilacqua, M. P., et al., Proc. Natl. Acad.Sci., USA 84:9238-9242 (1987). From these studies, it appeared thatELAM-1 may promote leukocyte adhesion by acting as a ligand for aleukocyte cell surface receptor. This hypothesis is further supported bythe present studies using cloned ELAM-1. Transfection of COS cells withpELAM-1 promotes the adhesion of HL-60 cells. Immunoperoxidase stainingrevealed a direct correlation between COS cell expression of ELAM-1 andHL-60 cell adhesion. Mab H18/7 was found to abate (100+3% inhibition,mean+SEM, 3 exp.), the augmented adhesion to pELAM transfected COScells.

Example 4 Expression of Natural ELAM-1

Previous studies have demonstrated that unstimulated HEC express littleor no ELAM-1. Bevilacqua, M. P., et al., Proc. Natl. Acad. Sci. (USA)84:9238-9242 (1987). Treatment of HEC monolayers for 4 hr with rIL-1 (5U/ml) or rTNF (200 U/ml) resulted in the production of anti-ELAM-1precipitable species. Similar results were obtained with lymphotoxin(200 U/ml) and bacterial endotoxin (10 ug/ml). No induction of ELAM-1was observed after treatment of the HEC with IFN (200 U/ml). RNA blotanalysis showed a primary species of 3.9 kb in HEC treated for 2 hr withrIL-1 or rTNF, that was absent in unstimulated and IFN-stimulated HEC.Continuous exposure of HEC monolayers to rIL-1 results in a rapidincrease in the expression of ELAM-1 with onset at 1-2 hr, and peak at4-6 hr, followed by a decline toward basal levels by 24 hrs. As shown inFIG. 2, endothelial cell surface expression of ELAM-1 , as detected inan immunobinding assay, correlates temporally with the expression ofELAM-1 mRNA. Thus, endothelial cell expression of ELAM-1 can be inducedby several mediators including rIL-1 and rTNF. These effects aretime-dependent and appear to occur, at least in part, at atranscriptional level. Blot hybridization of genomic DNA from placentarevealed a pattern consistent with a single-copy gene.

CONCLUSIONS

ELAM-1 is a novel cell surface glycoprotein with sequence homologies toa variety of complement binding proteins including complement receptor 1(CR1), complement receptor 2 (CR2), complement Factor H and C4 bindingprotein as well as some complement-binding proteins such as β₂-glycoprotein I. These homologies suggest the existence of a family ofrelated proteins. The translated amino acid sequences of ELAM-1 revealtypical features of a transmembrane protein including a leader sequence.The extracellular domain contains eleven potential N-linkedglycosylation sites and studies with endoglycosidase H suggest thatN-linked carbohydrates account for about 40% of the total molecular mass(Hr 115,000) of mature endothelial ELAM-1. In addition, these studies,in conjunction with pulse-chase labeling procedures, provide strongevidence that the two polypeptides immunoprecipitated from activated HECby anti-ELAM-1 Mabs are composed of the same polypeptide backbone; thedifference in molecular mass between the two species appears to involvepost-translational modification, probably at the level of carbohydrateprocessing.

The inducible expression of ELAM-1 may be of central importance in itsrole in leukocyte-vessel wall interactions. The data indicates thatcertain inflammatory/immune cytokines including IL-1 and TNF, but notIFN-, act on endothelial cells to induce the biosynthesis and expressionof ELAM-1. RNA blot analysis suggests that the induction of ELAM-1occurs, in large part, at the transcriptional level. Previous studieswith Mab H4/18 have demonstrated focal endothelial expression of ELAM-1in a variety of pathophysiological settings.

Having now fully described this invention, it will be understood bythose of skill in the art that the same can be performed within a wideand equivalent range of conditions, formulations, and other parameterswithout affecting the scope of the invention or any embodiment thereof.

What is claimed is:
 1. A method for detecting ELAM-1 expression in apatient, comprising (i) administering to said patient for a time, andunder conditions sufficient for binding, a pharmaceutical compositioncomprising a detectable amount of a labelled anti-ELAM-1 H18/7monoclonal antibody, or a labelled antibody fragment comprising an Fabdomain thereof, and a pharmaceutically acceptable carrier, and (ii)detecting said label.